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The development of biochemical analysis techniques to study heterogeneous biological samples is increasing. These techniques include synchrotron radiation Fourier transform infrared (SR-FTIR) microspectroscopy. This method has been applied to analyze biological tissue with multivariate statistical analysis to classify the components revealed by the spectral data. This study aims to compare the efficiencies of SR-FTIR microspectroscopy and focal plane array (FPA)-FTIR microspectroscopy when classifying rice tissue components. Spectral data were acquired for mapping the same sample areas from both techniques. Principal component analysis and cluster imaging were used to investigate the biochemical variations of the tissue types. The classification was based on the functional groups of pectin, protein, and polysaccharide. Four layers from SR-FTIR microspectroscopy including pericarp, aleurone layer, sub-aleurone layer, and endosperm were classified using cluster imaging, while FPA-FTIR microspectroscopy could classify only three layers of pericarp, aleurone layer, and endosperm. Moreover, SR-FTIR microspectroscopy increased the image contrast of the biochemical distribution in rice tissue more efficiently than FPA-FTIR microspectroscopy. We have demonstrated the capability of the high-resolution synchrotron technique and its ability to clarify small structures in rice tissue. The use of this technique might increase in future studies of tissue characterization.

Ban Chiang () is an extensively studied archeological site in Northeast Thailand, Udon Thani Province, which became a UNESCO world heritage in 1992. Depending on their production period ceramic artefacts show characteristic patterns at the surface which may be interpreted as iconographic motifs for the site. Two ceramic samples, excavated in 2003, from different periods were re-investigated, previously studied by Tanthanuch W., Pattanasiriwisawa W., Somphon W., Srilomsak S. Synchrotron studies of Ban Chiang ancient pottery. Suranaree J. Sci Technol. 2011, 18, 15–28 who focussed on thermal firing and redox-conditions. Shards selected for this study were sample 5412-S6E15 dated from Bronze Age (ca. 1000–300 BC) with yellow-brown, paint-free surface and a younger sample 8027 from Iron Age (ca. 300 BC–200 AD) with red painted design carrying pictorial patterns typical for the Ban Chiang ceramics. The surface material and colour of both shards was studied in detail using optical reflectance, FTIR spectroscopy and X-ray powder diffraction. Thin sections were used to determine the thickness of the red paint and the elemental composition of the surface using an electron microprobe. Chemical composition of sample 5412-S6E15 consists as oxides of elements mainly of SiO2 and Al2O3 (representing ca. 59 and 20 wt. %) and that of the red colour of the youngest sample 8027 consists mainly of SiO2, Al2O3 and Fe2O3 (representing ca. 52, 13 and 13 wt. %), the average composition adjacent to the red painted area has ca. 72 wt. % SiO2, ca. 10 wt. % Al2O3 and only 2 wt. % Fe2O3. X-ray powder diffraction, FTIR spectroscopy and optical reflectance measurements confirm hematite in the mineralogical composition of the red pigment of shard 8027.

Lindernia crustacea (L.) F. Muell. var. crustacean or “Ya Kap Hoi: YKH”, which is an edible vegetable, is commonly seen in Thailand. This study investigated its anticancer properties with high antioxidant activity by reducing power activity, excluding alkylation activity. It explored YKH extract induction of anticancer activity through biomolecular changes in the HCT116 human colon cancer cell line. The ethanolic extract stock solution and water extract stock solution were prepared. NR assay was used for cancer cell cytotoxic testing and Fourier transform infrared (FTIR) microspectroscopy was used for biomolecular changes study on lipid, protein and nucleic acid/DNA. The apoptosis induction by the extracts was detected by using Annexin V-FITC and DAPI staining. The compounds in the YKH ethanolic extract was performed by using GC-MS analysis. As a result, YKH ethanolic extract caused HCT116 colon cancer cell death in the dose dependent manner after 24 h exposure, and the 50 % cell death (IC50 concentration) was 195.4 ± 12 μg/mL. Cellular biochemical changes observed from FTIR data showed that YKH ethanolic extract treated HCT116 colon cancer cell. There is an increase in lipid content and a reduction in intensity of nucleic acid/DNA, a-helix protein structure at 1,656 cm-1 was reduced and peak position of b-sheet structure (1,637 cm-1) was shifted to lower frequency. From the analysis results, YKH ethanolic extract seems to exert anti-colon cancer effect by changing cellular biomolecular structure of lipids and β-sheet protein accumulation, supporting apoptotic induction. The compounds in the YKH ethanolic extract mainly yielded fatty acids,which may be useful as potential compounds.

Chaetoceros sp. has been found to possess significant amounts of valuable fucoxanthin, phenolics, and flavonoids, with enormous nutraceutical and antioxidant potential. The aim of this study was to employ ultrasound-assisted extraction to determine the antioxidant properties of extracts prepared from Chaetoceros tenuissimus using different solvents: Propylene glycol (PG), acetone (AC), and methanol (ME), with difference ultrasonication times (15 and 30 min). The total phenolic (TPC), flavonoid (TFC), and fucoxanthin (FUC) contents were evaluated. Antioxidant capacity was evaluated by DPPH and ABTS radical-scavenging assays and lipid peroxidation by TBARS. The FUC content was analyzed by HPLC-DAD. 30-min PG extract (PG-30) exhibited the highest TPC (2.75 ± 0.14 mg GAE.g−1 DW), and FUC (8.76 ± 0.06 mg FUC. g−1). Concerning total antioxidant capacity, PG-30 was the most effective against DPPH• (IC50 = 970.19 ± 7.70 µg. mL−1) and TBARS (IC50 = 717.05 ± 1.45 µg. mL−1), while 15-min PG extract (PG-15) presented the highest inhibitory activity against ABTS+• (IC50 = 150.02 ± 6.95 µg. mL−1). The study findings demonstrate that PG extracts of C. tenuissimus exhibit high potential antioxidant activity, likely associated with their content of FUC and phenolics, and could be used in cosmetic preparations and as phytonutrients. HIGHLIGHTS Chaetoceros tenuissimus Meunier from Chonburi province, Thailand High antioxidant activities of tenuissimus extracts by ultrasound assisted extraction with different solvents The correlation between chemical substances content and antioxidant activity GRAPHICAL ABSTRACT

Objective: To investigate biomolecular alteration of sesamol on human lung adenocarcinoma (SK-LU-1) cells compared with cisplatin using Fourier transform infrared microscopy (FTIR). Methods: Cytotoxicity of sesamol was investigated against SK-LU-1 cells by using neutral red. DNA fragmentation and the cell cycle analysis were determined by agarose gel electrophoresis and flow cytometry, respectively. The FTIR microscopy technique was applied to explore the changes in cellular biochemical compositions in cells treated with sesamol that the biochemical and biological assays cannot cover. The alkylating property was determined by 4-(4-nitrobenzyl)pyridine assay. Results: Sesamol and cisplatin exerted an antiproliferative effect at 48 h with respective IC50 values of 2.7 and 0.07 mM. Both induced apoptosis by causing DNA damage and accumulation of cell populations at sub-G1. FTIR microscopy and Principle Component Analysis clearly discriminated the sesamol- and cisplatin-treated cells from the untreated cells or control. A significant increase of total lipid content was found in cisplatin-treated cells. Conformational changes in the proteins secondary structure from the α -helix to the β -sheet were found in both sesamol- and cisplatin-treated cells, as well as significant reductions in relative DNA content of both compared to the control were observed, suggesting DNA damage. A shift in the peak position of DNA region provides insight on the DNA interactions. Conclusions: The non-alkylating effect of sesamol based on the nitrobenzyl pyridine assay delineates the non-covalent binding mode of sesamol on DNA. Hydrogen bonding is the binding mode of sesamol on DNA, while for cisplatin it was covalent and hydrogen bonding.

Lindernia crustacea (L.) F. Muell. var. crustacean or “Ya Kap Hoi: YKH” was misunderstood as “Ya Yad Nam Kang” and cancer curative. This study aimed to investigate anticancer activity by the analysis of chemical constituent in extracts and in vitro screening of biological activities of the extract in several aspects such as cancer cell lines cytotoxic activity, immune cell proliferating activity, reducing power activity, alkylation activity. The HPLC analysis showed the absence of plumbagin peak observed in the HPLC chromatograms of both YKH aqueous and YKH ethanolic extracts. The YKH ethanolic extract yielded more chemical constituents than that of the YKH aqueous extract. The YKH aqueous extract was inactive against all cancer cells tested. Interestingly, YKH ethanolic extract caused cancer cell death in HCT116 colon cancer, HepG2 liver cancer, and Jurkat leukemic cancer cell lines in the concentration dependent manner. The following IC50 concentrations of the YKH ethanolic extract that possessed 50 % cell death after 24 h exposure in HCT116, HepG2, and Jurkat cell line were 195.4 ± 12, 171.7 ± 8.7, and 48.8 ± 5.7 µg/mL, respectively. Both aqueous and ethanolic extracts of YKH showed high antioxidant activity based on reducing power activity but did not have alkylation activity. At high concentration (250 µg/mL), YKH ethanolic extract can inhibit immune cells proliferation activity more than the YKH aqueous extract. An unexpected but critical outcome of our studies was the finding that anticancer activity is promised by selecting the plant extraction solvent with less polarity. Potential anticancer constituents were extracted from YKH using ethanol and these constituents cannot be found in aqueous solution of YKH.

Sinorhizobium sp. BL3 forms symbiotic interactions with mung bean (Vigna radiata) and contains lrpL-acdS genes, which encode the 1-aminocyclopropane-1-carboxylate (ACC) deaminase enzyme that cleaves ACC, a precursor of plant ethylene synthesis. Since ethylene interferes with nodule formation in some legumes and plays a role in senescence in plant cells, BL3-enhancing ACC deaminase activity (BL3(+)) and defective mutant (BL3(-)) strains were constructed in order to investigate the effects of this enzyme on symbiosis and nodule senescence. Nodulation competitiveness was weaker in BL3(-) than in the wild-type, but was stronger in BL3(+). The inoculation of BL3(-) into mung bean resulted in less plant growth, a lower nodule dry weight, and smaller nodule number than those in the wild-type, whereas the inoculation of BL3(+) had no marked effects. However, similar nitrogenase activity was observed with all treatments; it was strongly detected 3 weeks after the inoculation and gradually declined with time, indicating senescence. The rate of plant nodulation by BL3(+) increased in a time-dependent manner. Nodules occupied by BL3(-) formed smaller symbiosomes, and bacteroid degradation was more prominent than that in the wild-type 7 weeks after the inoculation. Changes in biochemical molecules during nodulation were tracked by Fourier Transform Infrared (FT-IR) microspectroscopy, and the results obtained confirmed that aging processes differed in nodules occupied by BL3 and BL3(-). This is the first study to show the possible role of ACC deaminase activity in senescence in determinate nodules. Our results suggest that an increase in ACC deaminase activity in this strain does not extend the lifespan of nodules, whereas the lack of this activity may accelerate nodule senescence.

Objectives Anti-cancer effects of melatonin (N-acetyl-5-methoxytryptamine, an indole-amine), have been widely reported, however, little has been known, regarding its mechanism(s) of action in lung cancer. Thus, we investigated its induction of apoptosis through biomolecular changes (lipid, protein and nucleic acid/DNA) in the SK-LU-1 human lung cancer cell line. Materials and methods We used Fourier transform infrared (FTIR) microspectroscopy, and conventional methods, to confirm changes in lipid (annexin V/PI staining for membrane alteration), protein (caspase-3/7 protein activity) and DNA (DAPI staining for DNA fragmentation). Results We observed from FTIR data that melatonin increased lipid content and reduced intensity of nucleic acid/DNA, confirmed by annexin V/PI and DAPI respectively. Secondary protein structure at 1656 cm−1 (α-helix) was reduced and peak position of β-sheet structure (1637 cm−1) was shifted to lower frequency. Alteration in apoptotic proteins was demonstrated via caspase-3/7 activity induction. Conclusions High melatonin concentration exerted anti-cancer effects by changing biomolecular structure of lipids, nucleic acids and proteins, supporting its enhancement of apoptotic induction.

Dan Kwian, Sukhothai and Ratchaburi pottery clays are economically important pottery clays. They are well known in the Thai ceramic society for making Dan Kwian pottery, Sawankhalok pottery and Dragon jars, respectively. There have been several studies of these pottery clays. However, few of them used statistics to analyze their results. This work is a comparative study of the compositions and properties of these three pottery clays using statistical software to analyze the results. Results show that the major components of these pottery clays are SiO2 and Al2O3. The Modulus of Rupture (MOR) of each pottery clay fired at 700°C are not significantly different. Sukhothai pottery clay fired at 900°C has a higher MOR and bulk density while its porosity, water absorption and apparent specific gravity are less than those of the Dan Kwian and Ratchaburi pottery clays fired at the same temperature. Additionally, the current study developed regression equations for estimating the properties of all pottery clays under study. Finally, it was found that the L*, a*, b* and reflectance spectra of all pottery clays increased when firing temperature was increased from 700 to 900°C.

FTIR microspectroscopy was applied for studying macromolecular changes in human serum samples from patients with healthy livers, and those diagnosed with liver cirrhosis or hepatocellular carcinoma (HCC). Our study demonstrated that the serum samples from HCC and cirrhotic patients could readily be discriminated from those from healthy controls based on macromolecular differences related to their lipid and protein structure. Spectral changes appeared to indicate that the secondary structure of protein from HCC sample groups contained a more distinctive β -sheet structure and a lower lipid content compared to samples from the healthy and cirrhosis group. This was correlated with measurements of large decreases in albumin levels in serum from diseased patients. We argue that this technique shows potential as a simple, rapid, inexpensive, and non-subjective methodology for the screening patients suspected of liver disease. (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Molecular Dynamics (MD) simulation and the extended X-ray absorption fine structure (EXAFS) spectroscopy were employed to obtain the atomistic solvation structure of calcium (Ca2 +) ion in poly(vinyl alcohol) (PVA). From MD simulation results, it is obvious that there is only one dominant shell from oxygen atoms around calcium ion. The distance between the calcium ion and oxygen atoms in the first shell is in good agreement with experiment within 0.1 A. For the Ca K-edge EXAFS spectrum of CaCl2/PVA sample, there is no evidence for the formation of significant numbers of Ca2 +–Cl− contact ion pairs. Ca2 + ion can induce the conformational changes of PVA backbone compared to those of neat PVA. The MD and EXAFS technique is a powerful method to elucidate the detailed solvation structure of the probed ion in polymer matrix at the atomistic scale.

We evaluated the feasibility of FTIR microspectroscopy combined with partial least squares regression (PLS-R) for determination of resistance in HepG2 cells. Cell viability testing was performed using neutral red assay for the concentration of cisplatin resulting in 50% antiproliferation (IC50). The resistance index (RI) is the ratio of the IC50 in resistant HepG2 cells vs. parental HepG2 cells. Principal component and unsupervised hierarchical cluster analyses were applied and a differentiation of samples of cells (parental, 1.8RI, 2.3RI, 3.0RI, and 3.5RI) was demonstrated (3000-2800cm-1 in the lipid and 1700-1500cm-1 in the protein regions. The FTIR spectra were preprocessed with several treatments to test the algorithm. PLS-R models were built using the 1170 spectra of the HepG2 cells. Cross-validation was used to evaluate prediction of the RI value using this model. PLS-R models-preprocessed with the second derivative FTIR spectra-yielded the best model (R2=0.99, RMSEE=0.095 and RPD=7.98). Most RI values were predicted with high accuracy (91-100%) such that the linear correlation between the actual and predicted RI values was nearly perfect (slope~1). FTIR microspectroscopy combined with chemometric analysis using PLS-R offers quick, accurate, and reliable quantitative analysis of HepG2 cell resistance.

The first microbeam synchrotron X-ray fluorescence (µ-SXRF) beamline using continuous synchrotron radiation from Siam Photon Source has been constructed and commissioned as of August 2011. Utilizing an X-ray capillary half-lens allows synchrotron radiation from a 1.4 T bending magnet of the 1.2 GeV electron storage ring to be focused from a few millimeters-sized beam to a micrometer-sized beam. This beamline was originally designed for deep X-ray lithography (DXL) and was one of the first two operational beamlines at this facility. A modification has been carried out to the beamline in order to additionally enable µ-SXRF and synchrotron X-ray powder diffraction (SXPD). Modifications included the installation of a new chamber housing a Si(111) crystal to extract 8 keV synchrotron radiation from the white X-ray beam (for SXPD), a fixed aperture and three gate valves. Two end-stations incorporating optics and detectors for µ-SXRF and SXPD have then been installed immediately upstream of the DXL station, with the three techniques sharing available beam time. The µ-SXRF station utilizes a polycapillary half-lens for X-ray focusing. This optic focuses X-ray white beam from 5 mm × 2 mm (H × V) at the entrance of the lens down to a diameter of 100 µm FWHM measured at a sample position 22 mm (lens focal point) downstream of the lens exit. The end-station also incorporates an XYZ motorized sample holder with 25 mm travel per axis, a 5× ZEISS microscope objective with 5 mm × 5 mm field of view coupled to a CCD camera looking to the sample, and an AMPTEK single-element Si (PIN) solid-state detector for fluorescence detection. A graphic user interface data acquisition program using the LabVIEW platform has also been developed in-house to generate a series of single-column data which are compatible with available XRF data-processing software. Finally, to test the performance of the µ-SXRF beamline, an elemental surface profile has been obtained for a piece of ancient pottery from the Ban Chiang archaeological site, a UNESCO heritage site. It was found that the newly constructed µ-SXRF technique was able to clearly distinguish the distribution of different elements on the specimen.

Embryonic Stem-derived Neural Cells (ESNCs) hold potential as a source of neurons for a cell-based therapy for the treatment of brain tumors, and other neurological diseases and disorders in the future. The sorting of neural cell types is envisaged to be one of the most important processed for clinical application of these cells in cell-based therapies of the central nervous system (CNS). In this study, laboratory-based FTIR and Synchrotron-FTIR (SR-FTIR) microspectroscopy were used to identify FTIR marker for distinguishing different neural cell types derived from the differentiation of mouse embryonic stem cells (mESCs). Principal Component Analysis (PCA) and Unsupervised Hierarchical Cluster Analysis (UHCA) were shown to be able to distinguish the developmental stage of mESCs into three cell types: embryoid bodies (EBs), neural progenitor cells (NPCs), and ESNCs. Moreover, PCA provided the mean for identifying potential FTIR “marker bands” that underwent dramatic changes during stem cell differentiation along neural lineages. These appeared to be associated with changes in lipids (bands from CH 2 and CH 3 stretching vibrations at ∼2959, 2923 and 2852 cm −1 ) and proteins (changes in the amide I band at ∼1659 and 1637 cm −1 ). The results suggested that lipid content of cells increased significantly over the time of differentiation, suggesting increased expression of glycerophospholipids. Changes in the amide I profile, suggested concomitant increases in α-helix rich proteins as mESCs differentiated towards ESNCs, with a corresponding decrease in β-sheet rich proteins, corresponding with changes in cytoskeleton protein which may have been taking place involved with the establishment of neural structure and function.

Synchrotron-Infrared (SR-IR) mapping and Focal plane array (FPA) imaging have been applied for discrimination of the three biochemical components of the mouse blastocyst. The mouse blastocyst consists of two clusters of cells known as the inner cell mass (ICM) formed within the blastocoel cavity and the thin layer of outer cells called the trophectoderm. Using Hierachical Cluster Analysis (HCA) and Principle Component Analysis (PCA), it can be shown that the composition and distribution of biochemical components within the blastocyst show differences in the protein secondary structure and the lipid content. It is worth noting that the secondary structure of the outer layer cells indicates more distinctive β-type secondary structure. The blastocoel cavity was observed to be predominantly α-helix. Significantly, the ICM region showed the predominant high absorption intensities of lipid content (CH2, CH3 symmetric and asymmetric stretching around 3000–2800 cm−1). The results show agreement between both SR-IR mapping and FPA-IR imaging. We propose that the biochemical difference within the blastocyst, especially the high lipid content in the ICM region, could be involved in the process of lipid signaling during pre-implantation. The use of both techniques is shown to be significant approach for revealing the biochemical components within the blastocyst.

Staining of germinating rice with X-Gal and immunohistochemical localization indicated that β- d -galactosidase (EC 3.2.1.23) is localized to the embryo, aleurone layer and surrounding tissues, and in the radicle and shoot after germination. The genes encoding two isoforms of β-galactosidase expressed in germinating rice and panicle, designated OsBGal1 and OsBGal2 , were cloned and sequenced. The full-length cDNA sequence of OsBGal1 encodes 843 amino acids, including a signal peptide of 25 residues. The related OsBGal2 clone from panicle at flowering stage encodes 715 amino acid residues, including a signal peptide of 20 residues. The two genes were found to be relatively highly expressed in seedling roots and shoots and in leaf sheath in 15–30-day-old plants, which contained major immunoreactive proteins of approximately 90 kDa for OsBGal1 protein and 55 kDa for OsBGal2 in extracts of these tissues. Both were also expressed at low levels in flowers and immature seeds, but only the OsBGal2 transcript was found in mature seeds. The predicted mature proteins of the rice β-galactosidases were expressed as thioredoxin fusion proteins in E. coli strain OrigamiB (DE3) to produce β-galactosidase activities not found in control extracts. Purified OsBGal1 fusion protein hydrolyzed and transglycosylated p- nitrophenyl ( p NP) β- d -galactopyranoside and hydrolyzed p NP β- d -fucopyranoside. Galactose was released by this enzyme from β-(1 → 3)-, β-(1 → 4)- and β-(1 → 6)-linked di- and trisaccharides of galactose and rice root and rice coleoptile alcohol-insoluble residue, but it did not hydrolyze larger [ 3 H]oligo-(1 → 4)-galactans (penta-, octa- and undecasaccharides), lactose, a galactosylated xyloglucan oligosaccharide (XLLG), total rice hemicellulosic polysaccharides or pectin.

Characterization and identification of cancer cell, chemotherapy, resistance is important for both routine cancer therapy and trouble-shooting the medication treatment regimen. Present techniques for characterizing cancer cell resistance require multiple methods and steps, which are time-consuming and expensive. We present a protocol for simple sample handling, rapid detection, and spectral characterization of early resistant hepatocellular carcinoma (HepG2) cells, using Fourier transform infrared microspectroscopy (FTIR). Studies on alteration of the biochemical properties in a resistant HepG2 cell were evaluated-viz., increase efflux proteins (MRP-1 and P-gp) activity, total GSH content, anti-apoptotic (Bcl2) expression, and reduction of pro-apoptotic (Bax) proteins. Principle component analysis (PCA) was used to discriminate resistant HepG2 cells from parental HepG2 cells. Three important FTIR spectral regions were evaluated for reproducibility and discrimination ability-viz., lipid (3,000-2,800 cm(-1)), protein (1,700-1,500 cm(-1)) and carbohydrate and nucleic acid (1,300-900 cm(-1)). These 3 spectral regions can be used as spectroscopic biomarkers for differentiation of early or low resistance. This work presents a novel concept for high-throughput, FTIR spectroscopic discrimination of early resistance; thus enabling identification and characterization of cancer cell resistance.

3, 21, Suranaree Journal of Science and Technology

FTIR microspectroscopy was applied for studying macromolecular changes in human serum samples from patients with healthy livers, and those diagnosed with liver cirrhosis or hepatocellular carcinoma (HCC). Our study demonstrated that the serum samples from HCC and cirrhotic patients could readily be discriminated from those from healthy controls based on macromolecular differences related to their lipid and protein structure. Spectral changes appeared to indicate that the secondary structure of protein from HCC sample groups contained a more distinctive β -sheet structure and a lower lipid content compared to samples from the healthy and cirrhosis group. This was correlated with measurements of large decreases in albumin levels in serum from diseased patients. We argue that this technique shows potential as a simple, rapid, inexpensive, and non-subjective methodology for the screening patients suspected of liver disease.

Background Hyperpigmentation is aesthetic undesirable. Sesamol and the standard antimelanogenic agent (kojic acid) were shown to hinder melanogenesis by blocking tyrosinase and reducing melanin content. Objective The FTIR microspectroscopy was used in an attempt to find a novel method to define biological alternation in a melanogenesis inhibition of sesamol and kojic acid. Methods Tyrosinase inhibition and melanin content of sesamol and kojic acid were evaluated. The FTIR microspectroscopy was adopted to define the vibrational characteristic involved with the melanogenesis in the untreated SK-MEL2 cells vs. the sesamol- and kojic-treated SK-MEL2 cells. Results Sesamol and kojic acid inhibited mushroom tyrosinase at IC 50 of 0.33 μg/ml and 6.1 ± 0.4 μg/ml, respectively. Moreover, 30 μg/ml sesamol inhibited 23.55 ± 8.25% cellular tyrosinase activity in human SK-MEL2 cells, while 600 μg/ml kojic acid inhibited 33.9 ± 1.4% cellular tyrosinase activity in the same cells. In the SK-MEL2-treated with two inhibitors, the FTIR spectra assigned to the lipid and nucleic acid bands were significantly depleted with the secondary protein structure shifted to a more β-pleated secondary protein one. Conclusion Both sesamol and kojic acid display a similar pattern of antimelanogenesis activity albeit to a different degree. The mechanism of their whitening effect may be via the alteration of (a) the enzyme conformation disallowing the ordinary enzyme–substrate interaction and maybe (b) the integrity of the lipid-containing melanosome. Our results support the alternative use of FTIR microspectroscopy as a simple and reagent-free method for characterization of biomolecular changes in human melanoma cells.

Functional hepatocytes differentiated in vitro from mesenchymal stem cells (MSCs) need to be fully characterized before they could be applied as a therapy to treat liver disease. Here, we employed Fourier Transform Infrared (FTIR) microspectroscopy to investigate the characteristics of hepatocyte-like cells derived from rat bone marrow mesenchymal stem cells (rBM-MSCs) by detecting changes in macromolecular composition occurring during the hepatogenesis process. Partial Least Squares Discriminant Analysis (PLS-DA) enabled us to discriminate undifferentiated rBM-MSCs, and early, mid-stage and late stage rBM-MSCs derived hepatocytes by their characteristic FTIR "spectroscopic signatures". The predominant spectroscopic changes responsible for this discrimination were changes in FTIR absorbance bands at: 3012 cm(-1) (cis C[double bond, length as m-dash]C stretch from unsaturated lipids), 2952 cm(-1) (ν(as)CH(3) from lipids), 2854 cm(-1) (ν(s)CH(2) from lipids) and 1722 cm(-1) (C[double bond, length as m-dash]O stretching from lipids), which were associated with triglyceride and unsaturated fatty acid accumulation in the hepatocyte-like cells occurring during differentiation. Based on these findings, rBM-MSCs derived hepatocytes are characterized by high lipid content which facilitates a means of identifying hepatocytes from their stem cells progenitors by using FTIR microspectroscopy. Other complex changes in spectral bands assigned to proteins and nucleic acids were observed during hepatocyte differentiation indicating that mRNA translation was taking place producing proteins related to the formation of the new hepatocyte-like phenotype, which was corroborated by immunohistochemistry. The results show FTIR microspectroscopy combined with bioinformatic modeling constitutes a powerful new phenotypic-based methodology for monitoring and characterization of the process of stem cell differentiation leading to the formation of hepatocytes, providing complementary information to existing methodologies such as immunohistochemistry and gene analysis, but having advantages of being reagent-free and non-destructive of the sample.

To evaluate the anticancer activity of the extract fraction of Polyalthia evecta (P. evecta) (Pierre) FinetGagnep and the synergistic anticancer effect of the extracts from P. evecta by using the ATR/FT-IR spectroscopy.The 50% ethanol-water crude leaf extract of P. evecta (EW-L) was prepared and was further fractionated to isolate various fractions. The anticancer activity was investigated from cytotoxicity against HepG2 using a neutral red assay and apoptosis induction by evaluation of nuclei morphological changes after DAPI staining. Synergistic anticancer effects of the extracts from P. evecta were performed using the ATR/FT-IR spectroscopy.The result showed that the EW-L showed higher cytotoxicity and apoptosis induction in HepG2 cells than its fractionated extracts. The hexane extract exhibited higher cytotoxicity and apoptosis induction than the water extracts, but less than the EW-L. The combined water and hexane extracts apparently increased cytotoxicity and apoptosis induction. The %apoptotic cells induced by the extract mixture were increased about 2-fold compared to the single hexane extract.The polar extract fraction is necessary for the anticancer activity of the non-polar extract fraction. The ATR/FT-IR spectra illustrates the physical interaction among the constituents in the extract mixture and reveals the presence of polyphenolic constituents in the EW-L, which might play a role for the synergistic anticancer effect.

Apoptosis is the principal molecular goal of chemotherapeutics for effective anticancer action. We studied the effect of 50% ethanolic-water extracts of Pinus kesiya, Cratoxylum formosum ssp. pruniflorum and melphalan on cytotoxicity and apoptosis induction for human leukemic U937 cells, and explored the mode of action using FTIR microspectroscopy. The number of viable U937 cells in vitro was decreased in a concentration-dependent manner by all tested compounds, although potency differed between the U937 and Vero cells. Melphalan and the extract of C. formosum exhibited relatively lower IC(50) values (15.0 ± 1.0 and 82.7 ± 3.2 μg/mL respectively) and higher selectivity (selective index3) than the extract of P. kesiya (299.0 ± 5.2 μg/mL; selective index3) on the U937 cells. All three compounds significantly induced apoptosis through the late stage - seen by the indicative DNA ladder - with the most effective being melphalan, then the P. kesiya and C. formosum extracts. FTIR microspectroscopy revealed that all three compounds raised the intensity of the β-pleated sheet - higher than that of the untreated U937 cells - corresponding to a shift in the α-helix band associated with an alteration in the secondary structure of the protein band, confirming induction of apoptosis via pro-apoptotic proteins. The differences in intensity of the FTIR bands associated with lipids, proteins and nucleic acids were responsible for discrimination of the anticancer mode of action of each of the three compounds. The FTIR data suggest that the two plant extracts possessed anticancer activity with a different mode of action than melphalan.

Background Many plant β-galactosidases (Bgals) have been well characterized and their deduced biological functions mainly involve degradation of structural pectins, xyloglucans or arabinogalactoproteins in plant cell walls. However, gene multiplicity in glycosyl hydrolase family 35 (GH35), to which these proteins belong, implies diverse functions. In this study, the gene multiplicity, apparent evolutionary relationships and transcript expression of rice Bgal genes were examined, in order to predict their biological functions. Results Fifteen rice Bgal genes were identified in the plant genome, one of which encodes a protein similar to animal Bgals (OsBgal9), and the remaining 14 fall in a nearly plant-specific subfamily of Bgals. The presence of both classes of Bgals in bryophytes, as well as vascular plants, suggests both gene lineages were present early in plant evolution. All 15 proteins were predicted to contain secretory signal sequences, suggesting they have secretory pathway or external roles. RT-PCR and database analysis found two distinct lineages to be expressed nearly exclusively in reproductive tissues and to be closely related to Arabidopsis Bgals expressed most highly in flower and pollen. On the other hand, OsBgal6 is expressed primarily in young vegetative tissues, and alternative splicing in panicle prevents its production of full-length protein in this reproductive tissue. OsBgal11 also showed alternative splicing to produce different length proteins. OsBgal13 produced by recombinant expression in Escherichia coli hydrolyzed α-L-arabinoside in addition to β-D-galactoside and β-(1→3)-, β-(1→4)- and β-(1→6)- linked galacto-oligosaccharides. Conclusion Rice GH35 contains fifteen genes with a diversity of protein sequences, predicted locations and expression and splicing patterns that suggest that OsBgals enzymes may play a variety of roles in metabolism of cell wall polysaccharides, glycoproteins and glycolipids.

Stem cell-based therapy for liver regeneration has been proposed to overcome the persistent shortage in the supply of suitable donor organs. A requirement for this to succeed is to find a rapid method to detect functional hepatocytes, differentiated from embryonic stem cells. We propose Fourier transform infrared (FTIR) microspectroscopy as a versatile method to identify the early and last stages of the differentiation process leading to the formation of hepatocytes. Using synchrotron-FTIR microspectroscopy, the means of identifying hepatocytes at the single-cell level is possible and explored. Principal component analysis and subsequent partial least-squares (PLS) discriminant analysis is applied to distinguish endoderm induction from hepatic progenitor cells and matured hepatocyte-like cells. The data are well modeled by PLS with endoderm induction, hepatic progenitor cells, and mature hepatocyte-like cells able to be discriminated with very high sensitivity and specificity. This method provides a practical tool to monitor endoderm induction and has the potential to be applied for quality control of cell differentiation leading to hepatocyte formation.